Project Summary Cellular migration is necessary for proper embryonic development as well as maintenance of adult health. Cells can migrate individually or in groups in a process known as collective cell migration. These cohorts of cells maintain cell-cell contact, group polarization and exhibit coordinated behavior. Collective cell migration is important in numerous processes during development including blood vessel branching and neural crest cell migration as well as in adulthood in wound healing. Additionally, this process is misappropriated during cancer cell invasion. During individual and collective cellular migration, cells must extend protrusions to interact with the extracellular environment, sense chemotactic cues, and act as points of attachment. The mechanisms and regulators of protrusive behavior have been widely studied in populations of cells that migrate individually; however, how protrusive behavior is regulated throughout collectives is not well understood. Specifically, it has been difficult to define protrusive behavior of cells inside the collective, as these cells are tightly packed. I am using the zebrafish posterior lateral line primordium (pLLP) as a model of collective cell migration in order to examine the regulation of protrusive behavior during collective cell migration. The pLLP is a cluster of around 100 cells that migrates along both lateral sides of the zebrafish, depositing groups of cells that will become sensory organs. These sensory organs make up the posterior lateral line (pLL), a mechanosensory system responsible for sensing changes in water current. I aim to use advantages of this system, including amenity to live imaging and genetic approaches, to define protrusive behavior throughout the pLLP and identify factors that regulate this behavior during collective cell migration. Towards this goal, I have performed mosaic analysis to sparsely label small numbers of cells in the pLLP with a transgene that marks filamentous actin. This approach revealed an abundance of brush-like actin-based protrusions in multiple cells across the pLLP. To identify genes that regulate protrusive behavior, I have examined expression of candidate genes, including those that are regulated by canonical Wnt signaling. Within the pLLP, canonical Wnt signaling is important for a number of cellular behaviors including migration. In the absence of Wnt signaling, the pLLP migrates abnormally, resulting in truncation of the pLL. We have found a number of Wnt target genes that regulate actin dynamics and thus might regulate protrusive behavior. Moreover, my preliminary data show that a subset of these genes is expressed in specific regions of the pLLP, and their expression pattern is perturbed under Wnt- deficient conditions. Based on this preliminary data, I will: define protrusive behavior throughout the pLLP and determine the role of Wnt signaling in regulating this behavior (Aim 1); and identify regulators of protrusive behavior during collective cell migration (Aim 2). Further elucidation of the regulation of protrusive behavior during collective cell migration could define new mechanisms of organ development as well as how these mechanisms are misappropriated during cancer cell invasion.